The Streptococcus sanguis Platelet Aggregation-associated Protein (PAAP) has been isolated as a precursor N-formylmethionyl glycoprotein of 115 kDa. While other prokaryotic glycoproteins have been suggested in the literature, this is the first protein from a gram positive organism, shown by direct experimental evidence to have covalently associated carbohydrate. Preliminary studies also suggest that the carbohydrate polymers are not present in the wall-associated form of PAAP. Therefore, by studying PAAP biosynthesis and export, S. sanguis provides a unique model system to study the novel biosynthetic pathway of prokaryotic glycoproteins. We hypothesize that PAAP provides a mechanism for incorporation of carbohydrate polymers onto the cell wall of S. sanguis. Acting as a cotransporter, PAAP may translocate the carbohydrate through the membrane in covalent association with the exported protein. Once PAAP is transported, the carbohydrate oligosaccharides may be cleaved from the protein backbone and transferred to other cell wall components, such as lipid or peptidoglycan. Therefore the long-term goal of our research is to delineate the biosynthetic pathway of cell wall-associated streptococcal glycoproteins. The specific aims of this research project are to isolate the S. sanguis glycosyltransferase and its gene. Future research would then delineate the glycosylation pathway (including other enzymes, protein consensus sequence for glycosylation, cation requirements, pH requirements, etc.), determine of whether glycosylation is cotranslational and/or a cotransport event, and compare this new glycosylation pathway with known eukaryotic pathways.